It is desirable to insert transgenes into adenoviruses for many reasons, for example, to arm therapeutic viruses to increase therapeutic impact or to deliver genes to target cells using a replication competent virus or a replication deficient viral vector.
Typically, to insert a transgene into a virus genome, a plasmid is generated comprising the adenoviral genome, the transgene is then inserted into the plasmid, for example employing homologous recombination and then the viral genome is excised from the plasmid. However, for reasons described herein, flexible plasmids that can be used for both replication competent viruses and replication deficient viral vectors and, for example which can accept large transgenes in a known and predictable location are not always readily available, especially if the transgene is to be inserted in an unusual location, such as outside the E1 or E3 region.
The problems associated with inserting transgenes into adenoviruses for therapeutic and diagnostic purposes fall into 3 main categories. Firstly, not all adenoviruses are ideal candidates for therapeutic and diagnostic applications, for example, Ad5 (a subgroup C adenovirus) immunity is prevalent in the human population and consequently the virus is rapidly cleared by the immune system after it is administered. To overcome this problem, adenoviruses to which there is less prevalent immunity have been utilised. However, much of the genomic work to date has been on Ad5. Therefore, the materials and resources for alternative adenoviruses are often not available.
Secondly, not all adenoviruses can accept large transgenes and maintain their stability as a viable viruses. Furthermore, the adenovirus genome is large and there is little room to insert additional genetic material without affecting a function of the virus, for example the function of packaging the virus into the viral capsid may be adversely affected, which in turn is likely to impact the infectivity of the virus.
To overcome this problem deletions have been made to the genome. This strategy is particularly suitable for replication deficient viral vectors because one or more genes are removed which are essential to replication. This both limits the vector's ability to replicate in vivo and creates space in the genome thereby allowing insertion of large transgenes. These transgenes can be expressed in vivo, regardless of the viral vector's inability to replicate. Most frequently the E1 gene has been deleted. Prior art systems, such as the ADEASY system (Agilent Technologies), allow insertion of transgenes to the E1 region. In some instances part, or all, of the E3 region is deleted, see for example WO2011/0123564 and the gene may be inserted in the region deleted.
Thus typically the transgene is inserted in the same position that the deletion occurred. Thus, the site of transgene insertion has largely been limited to the location of early genes which can be problematic because it is more likely to affect virus gene expression, virus life-cycle and/or speed of replication. In particular, deleting the E1 region is not appropriate for the replication competent adenoviruses and as discussed it may be useful to insert a transgene such that it is not in a location of an early gene to ensure that the impact on the virus life-cycle is minimised.
Thirdly, the adenovirus genome is not easy to manipulate because the genome is densely packed and has very little intergenic material where a transgene might be safely inserted without affecting the virus life-cycle and/or a function, such as transcription. Furthermore, there are few, if any, restriction sites in the intergenic regions and even fewer that only occur once in the genome. The latter is relevant because when a restriction site occurs more than once in the virus genome then the ability to selectively insert a transgene in one location employing that restriction site is severely impeded.
Therefore it is desirable to provide a plasmid that can be used to manipulate a replication competent virus and wherein transgenes may be inserted in a location removed from the early genes.
One strategy that can be utilised with replication competent viruses is to employ a non-biasedly inserting transposon to insert the transgene into the genome (as described in Jin et al 2004). The transposon may be inserted in the late genes and thus this technology does not suffer from the disadvantages of the systems described above. Jin et al hypothesised that the site of location of insertion of the gene is influenced by the type the gene being inserted and, whilst it was possible to replace some of the genes after insertion, in some instances this was difficult to replace the inserted gene or the replacement gene was inserted in a different orientation. The random nature of transposon insertion provides many possible insertion sites. Therefore, predictability and reproducibility of insertion may be compromised as a result. Furthermore the transposon inserts itself into the genome along with the transgene and in theory could “move” the location of the gene in the virus genome at a later date. However, whilst the randomly inserting transposon is a wonderful tool for investigating the virus genome the biggest disadvantage of this approach is that it does not allow rational design of the virus construct.
Therefore, it is desirable to provide a plasmid that can be used to manipulate a replication competent virus and in which transgenes can reproducibly be inserted in a location removed from the early genes. The present inventors set out to overcome one or more of the problems described above by generating a plasmid with a combination of restriction sites that can be used to selectively insert a transgene specifically into a location that this not the site of an early gene.
The present inventors have developed adenovirus plasmids comprising original restriction sites in the vicinity of the L5 gene. The plasmids of the present disclosure allow generation of viruses with restriction sites/transgenes in locations other than the early gene sites, for example for replication competent adenoviruses with the E1 region intact or replication deficient adenoviruses, such as with E1 and/or E3 deleted or interrupted.